In WO 97/13838 entitled Isolation of Cellular Material Under Microscopic Visualization published Apr. 17, 1997 the statement is made at page 20, line 24 the statement is made:                The size of the tissue transferred, depending upon the needs of the operator, can be varied by changing the diameter of the laser beam and pulse duration. Highly reproducible transfers in the 60 to 700 μm diameter range are easily attainable for procurement of small (100 μm to 1 mm) lesions without the encroachment of adjacent, non-neoplastic cells. In most basic and clinical research studies, procurement of several hundred to several thousand cells is necessary to provide sufficient genetic material for reliable amplification and statistically meaningful analysis. However, since laser beams can be focused to less than one cell diameter, transfers of targeted single cells or even parts thereof is thought possible under the practice of the invention.        
In the Application that follows we set forth the solution to the transfer that “is thought possible” mentioned above.
Although the first microdissection patents described a rigid inert substrate to which the thermoplastic polymer was applied which could be used as a pressure plate, the original implementation of LCM employed a freestanding film that was applied to the surface of the tissue by gently pressing the film onto the sample. The film above the tissue section of interest was then heated by a 100-micron diameter beam and melted by pulses from a CO2 laser. The length of the laser pulse (between 100 msec and 630 msec) was chosen so as to allow the irradiated film to come to a steady state temperature rise for sufficiently long times for the polymer to flow into the tissue and form a strong bond by replacing air voids within the desiccated sample. The 630 msec pulses typically used with this system were purposefully chosen to be that long to insure sufficient time after the steady state temperature was reached for the melted polymer (which remains molten until the end of the pulse) to reliably flow into the tissue during the laser pulse. In subsequent work it was shown that equivalent transfers could be achieved with this system and 100 msec pulses, although because of irregular spacing between the polymer surface and the tissue, transfer with 100 msec pulses were less reproducible than with the longer pulses. In practice with this LCM system, the objective was to heat the lower surface of the polymer to a little more than its melting point. The CO2 laser delivered power levels were kept within a factor of two of the threshold power required (range of 25-50 mW delivered to a 100 μm spot on an EVA polymer film 100 μm thick). Thus the tissue captured by the melted polymer was typically exposed to peak temperatures of ˜90-100 C for ˜500 msec. Using this process damage to DNA, RNA, or proteins in the captured sample was not observed by subsequent molecular analysis.
Short pulses were avoided in LCM so as to insure adequate bond strength. Information from a number of manufacturers of EVA-based thermoplastic adhesives (e.g., hot glue) suggested that using EVA adhesives required maintaining molten joint under pressure for more than one second. In the original CO2 laser LCM designs, the use of a pressure plate (transparent and non absorbing of the laser and visible light) was impractical because of the rarity and expense of materials that transmit CO2 laser wavelengths (9-11 μm). Subsequently the introduction of strongly-absorbing near infrared (˜0.8 μm) dyes soluble in the thermoplastic polymers allowed the transfer film to be focally melted by the pulsed infrared laser diodes (˜0.8 μm) easily focused through transparent substrates to small diameters less than 10 μm in the absorbing thermoplastic film.